Combining higher accumulation of amylopectin, lysine and tryptophan in maize hybrids through genomics-assisted stacking of waxy1 and opaque2 genes

Waxy maize rich in amylopectin has emerged as a preferred food. However, waxy maize is poor in lysine and tryptophan, deficiency of which cause severe health problems. So far, no waxy hybrid with high lysine and tryptophan has been developed and commercialized. Here, we combined recessive waxy1 (wx1) and opaque2 (o2) genes in the parental lines of four popular hybrids (HQPM1, HQPM4, HQPM5, and HQPM7) using genomics-assisted breeding. The gene-based markers, wx-2507F/RG and phi057 specific for wx1 and o2, respectively were successfully used to genotype BC1F1, BC2F1 and BC2F2 populations. Background selection with > 100 SSRs resulted in recovering > 94% of the recurrent parent genome. The reconstituted hybrids showed 1.4-fold increase in amylopectin (mean: 98.84%) compared to the original hybrids (mean: 72.45%). The reconstituted hybrids also showed 14.3% and 14.6% increase in lysine (mean: 0.384%) and tryptophan (mean: 0.102%), respectively over the original hybrids (lysine: 0.336%, tryptophan: 0.089%). Reconstituted hybrids also possessed similar grain yield (mean: 6248 kg/ha) with their original versions (mean: 6111 kg/ha). The waxy hybrids with high lysine and tryptophan assume great significance in alleviating malnutrition through sustainable and cost-effective means. This is the first report of development of lysine and tryptophan rich waxy hybrids using genomics-assisted selection.

Backcross-and self-progenies. The recurrent inbreds (as female) and donor inbred (as male) showing polymorphism for gene-based markers specific to both wx1 and o2 genes were crossed during the rainy season (July-November, 2016) at IARI, Delhi (28° 09′ N, 77° 13′ E, 229 MSL). F 1 s were grown during the winter season (December, 2016-April, 2017) at IIMR-Winter Nursery Centre (WNC), Hyderabad (17° 19′ N, 78° 25′ E, 542.6 MSL). BC 1 F 1 progenies were grown at Delhi during the rainy season (2017), and foreground selection was carried out using the wx1 and o2 specific markers. The foreground positive plants along with high recovery of the recurrent parent genome (RPG), maximum phenotypic similarity to recurrent parents and endosperm opaqueness of 25-50% were backcrossed to the respective recurrent parents 15 . The BC 2 F 1 populations raised at Hyderabad during winter season (2017-2018), and were subjected to foreground-, background-and phenotypic selection were carried out. The foreground positive plants with a maximum RPG, morphological similarity and similar kernel opaqueness (25-50%) to their recurrent parents were selfed. The BC 2 F 2 progenies were grown during the rainy season (2018) at Delhi. Foreground positive plants homozygous for wx1 and o2 gene were subjected to the background-and phenotypic-selection including the kernel modification. The selected plants were self-pollinated to generate BC 2 F 3 progenies during rainy season (2019) at Delhi (Table S3). White kernel progenies with 25-50% opaqueness in endosperm were chosen in each of the three genetic backgrounds. In all the BC 1 F 1 , BC 2 F 1 and BC 2 F 2 generations, kernels with 75-100% opaqueness were not considered 15 . The details of backcross-and self-generations grown at different locations and seasons are described in Table S3, while marker-assisted backcross breeding (MABB) scheme 22,23 followed in the present study is represented in Fig. 1.

DNA isolation and polymerase chain reaction amplification and electrophoresis. The CTAB
method was used to isolate genomic DNA from young seedlings (3-4 leaf stage) 24 . Polymerase chain reaction (PCR) amplification and electrophoresis of the PCR products for the wx1 and o2 genes were performed using protocol standardized at Maize Genetics Unit, IARI 4,22 . PCR was performed in 20 μl volume on Veriti 96-well thermal cycler (M/s. Applied Biosystems) using GeneDirex OnePCR reaction mixture. Amplification of PCR products was performed with a 'touch-down 60' procedure as per Duo et al. 1 Electrophoretic separation of the PCR products was performed using 4% agarose (Lonza, Rockland, ME USA) at 100-120 V for 3-4 h with a 50 bp DNA ladder (MBA-Fermentas). Photographs of the amplified products was captured using gel documentation system (AlphaInnotech, California, USA).

Marker-assisted foreground selection for wx1 and o2 gene
Hybridity testing was undertaken in F 1 s using markers specific to wx1 and o2 genes. Foreground selection was performed in BC 1 F 1 , BC 2 F 1 , and BC 2 F 2 generations. Gene based InDel marker, wx-2507F/RG was used for selection of wx1 gene 25 . Heterozygous plants (Wx1/wx1) were selected in the BC 1 F 1 and BC 2 F 1 , while homozygotes (wx1/wx1) were selected in BC 2 F 2 . SSR, phi057 was used to genotype the populations and homozygotes (o2o2) were selected in BC 1 F 1 26 . The details information of markers used in foreground selection are presented in Table S4.

Marker-assisted background selection for recurrent parent genome
A set of > 320 genome-wide SSRs covering all the 10 chromosomes of the maize genome were used for identifying polymorphic markers between the respective recurrent and donor parents ( Table 1). The sequence of SSR primers was retrieved from the maize genome database (www. maize gdb. org) and was custom synthesized (Sigma Tech., USA). PCR amplification and scoring of amplicons of SSRs employed in background selection were carried out as per Hossain et al. 22 Polymorphic SSRs between the recurrent and donor parents were used to recover the RPG in individuals from the BC 1 F 1 , BC 2 F 1 and BC 2 F 2 populations.
Agronomic evaluation of MABB-derived inbreds. MABB-derived inbreds (three from each of the four genetic background) and their recurrent parents were evaluated in randomized complete block design (RCBD) with two replications at the IARI, Delhi during the rainy season (2020). Each inbred was grown in a 3 m row, with a 75 cm row-to-row and 20 cm plant-to-plant distance. Inbreds were characterized for five important agronomic traits [days to 50% anthesis (MF), days to 50% silking (FF), plant height (PH), ear height (EH) and grain yield (GY)] and 31 morphological characters pertaining to distinctness, uniformity and stability (DUS) 27 . Standard agronomic practices were followed to raise the good crop. Two to three plants per entry were self-pollinated to avoid any xenia effects caused by foreign pollens, and the selfed grains were analyzed for amylopectin, lysine and tryptophan. Characters namely MF, FF, PH, EH and GY were recorded from open pollinated plants.
Agronomic evaluation of reconstituted hybrids. Selected three BC 2 F 3 progenies from each of the four inbreds were used to reconstitute 12 F 1 hybrids during the winter season (2019-20) at Hyderabad. Three versions of the reconstituted hybrids (-A, -B, and -C) and their corresponding original hybrid in each of the four hybrid  Analysis of amylopectin. Self-pollinated grains were used to estimate amylopectin from maize kernels.
Absolute amylose content was estimated as per Gibbon et al. 28 with minor modifications. Around 8-10 dried maize seeds were ground into seed powder with a diameter of < 0.2 mm using seed grinder (Cyclotec Sample Mill-1093, Sweden). Weighted 100 mg of seed powder was treated with 500 µl of 80% ethanol and vortexed for a short time. The sample tubes were centrifuged for 5 min at 10,000 rpm and supernatant was separated. The residues of the samples were again treated with 10% toluene and centrifuged for 5 min at 10,000 rpm and supernatant was separated. The process was repeated until the supernatant was clear of white layer. The supernatant was discarded, and the residue was fully dried in an incubator at 80 °C for 3-4 h. The resulting residue represented starch with a < 5% impurity level. 25 mg of the starch residue was placed into a 50 ml falconer tube. It was solubilized with 2.5 ml 1 M NaOH and mixed properly, and heated for 20 min in a hot water bath at 80 °C. The volume was adjusted to 25 ml with double distilled water after the samples were cooled to room temperature. 1.25 ml samples were transferred from the above sample into a new 50 ml falconer tube and treated with 125 µl 1 N acetic acid, 100 µl 1 M NaOH, and 500 µl of I 2 -KI solution. The samples were incubated at room temperature for 20 min to generate colour, then measured at 620 nm for absorbance (G-Biosciences Spectrophotometer, BT-UVS-SBA-E, BenchTop). The percent of amylose was calculated using the average of three technical replicates. The percent amylopectin was obtained by subtracting amylose from 100.
Analysis of lysine, and tryptophan. The lysine and tryptophan of maize kernels was estimated using UHPLC (Dionex Ultimate 3000 System, Thermo Scientific, Massachusetts, USA). The selfed seeds were dried and ground into powder, and further used for estimation of lysine and tryptophan 29 . The flour of the grains was acid hydrolyzed using 800 μl of 6 N HCl, 100 μl of 0.1 N HCl, 100 μl of nor-leucine and 10 μl of phenol for 16 h at 110 °C. Two mobile phases, A and B consisted of buffer and organic phase in the ratio of 9:1 (v/v) and 1:9 (v/v), respectively were used for estimation of lysine. Buffer phase for lysine contained tetra-methyl ammonium chloride and sodium acetate trihydrate (pH 3.5), while organic phase had acetonitrile and methanol (49:1, v/v). In case of tryptophan, alkaline hydrolysis (2 ml of 4 M NaOH and 200 μl of 0.1% ascorbic acid for 16 h at 110 °C) was performed. The mobile phase for tryptophan consisted of water and acetonitrile in the ratio of 95:5. The samples were injected separately in UHPLC through Acclaim 120 C 18 column (5 μm, 120 Å, 4.6 × 150 mm) with a flow rate of 1.0 and 0.7 ml/min, and detected using RS 3000 photodiode array (PDA) detector at 265 and 280 nm, respectively. The concentration of lysine and tryptophan was estimated in three technical replicates by standard regression curve derived using dilutions of external standards (AAS 18-5ML, Sigma Aldrich).

Statistical analysis.
Chi-square analysis was used to test the goodness of fit of the observed segregation pattern of wx1 across segregating populations (BC 1 F 1 , BC 2 F 1 and BC 2 F 2 ), as well as o2 in the BC 1 F 1 generation 22 .
The amplicons of SSRs used in background selection were scored as "A" for the recurrent parent, "B" for the donor parent, and "H" for the heterozygous genotype. Recovery of RPG was estimated using formula 30   www.nature.com/scientificreports/ recovery of RPG in selected backcross-derived progenies 31 . Graphical representations on amylopectin, lysine and tryptophan in each genotype were made using Microsoft Excel (2013). Windostat v10 software was used to analyze the agronomic and biochemical data.

Research involving plants.
No approvals were required for the study, which complied with all relevant regulations.

Results
Marker polymorphism among parents. Gene-based InDel marker, wx-2507F/RG was polymorphic between recurrent (HKI161, HKI163, HKI193-1 and HKI193-2) and donor (MGU-102-wx1) parents. wx-2507F/RG amplified 280 bp fragment in all the four recurrent inbreds, while it amplified 260 bp fragment in waxy donor line ( Fig. 2A). Gene-based SSR, phi057 produced 165 bp allele in all four recurrent parents, while the donor generated 153 bp allele (Fig. 2B). A range of 102-273 plants across BC 1 F 1 , BC 2 F 1 and BC 2 F 2 were subjected to foreground selection using wx1 and o2 gene ( Genomics-assisted selection.    genotype were selected for white colour in kernels. Seeds with yellow kernel colour were not considered for the present study. The white seeds homozygous for both wx1 and o2 genes were planted in order to generate BC 2 F 3 progenies. Three progenies each in HKI161-, HKI1163-, HKI193-1-and HKI193-2-based populations were finally selected for evaluation and reconstitution of hybrids ( Table 4).  www.nature.com/scientificreports/ age of ~ 1.4-fold increase in amylopectin was recorded among introgressed progenies. However, starch content among introgressed-(mean: 69.42%) and original-inbreds (mean: 68.05%) were statistically at par (Table S5).

Discussion
Waxy maize rich in amylopectin is highly popular in East and Southeast Asia 4,32 . Though large number of waxy maize cultivars are available for commercial cultivation worldwide 4 , waxy maize protein is poor in nutritional quality due to sub-optimal levels of essential amino acids like lysine and tryptophan 6,13 . Lack of waxy hybrids rich in lysine and tryptophan limits its great potential as a nutritious food to the resource poor especially in the developing countries 3 . Here, we used genomics-assisted breeding to combine high amylopectin, lysine and tryptophan in the genetic background of four popular sub-tropically adapted hybrids through marker-aided selection of recessive wx1 and o2 genes. The gene-based markers viz., wx-2507F/RG and phi057 helped in precisely selecting individual plants with favourable allele of both wx1 and o2 genes, respectively. Both the markers behaved co-dominantly and distinguished the homozygotes from heterozygotes 33 . Hossain et al. 3 reported polymorphism among Wx1 and wx1 alleles using wx-2507F/RG. Zhang et al. 11 observed polymorphism in wx1 gene among recurrent and donor parents using gene-based SSRs viz., phi027, phi061, and phi022. While, Yang et al. 13 reported phi022 and phi027 as polymorphic among the recurrent and donor parents. Several authors have also successfully used gene-based SSRs, phi057 and umc1066 to select o2 allele in the MABB programme 22,26 . Identification of heterozygotes (BC 1 F 1 and BC 2 F 1 ) and homozygotes (BC 2 F 2 ) at seedling stage helped in the exclusion of non-target progenies, resulting in significant savings of labour and material cost required for raising crops and pollination activities 29,34 . In the present study, wx1 gene segregated as per Mendelian ratio of 1:1 in backcross generations and 1:2:1 in selfed generations. Yang et al. 13 also reported 1:1 segregation in BC 1 F 1 and BC 2 F 1 , while reported 1:2:1 ratio in F 2 populations segregating for wx1 gene. However, segregation distortion (SD) was observed for o2 gene in some crosses. Similar observation was also observed by Jompuk et al. 35 and Hossain et al. 22 while analyzing the segregation of o2 in various backcross populations. This SD could be caused by gametophytic factors, mutants such as faulty kernels, male sterility, and embryo-specific mutations 36 . SD warrants raising of large population size in order to achieve sufficient foreground positive genotypes in the MABB programme.
Since, o2 and wx1 genes are recessive, traditional backcross approach would have taken 12-14 seasons as each backcross generation would require progeny testing by selfing 22 . Two generation-based MABB, on the other hand, was efficient enough to generate comparable results in nearly half of the time (5-6 seasons). MABB strategy thus saved significant time and resources besides speeding up the breeding cycle 37 . Genomics-assisted background selection achieved high recovery of RPG in just two backcross generations 11,22 . The high recovery of RPG resulted in the phenotypic resemblance with their original versions. The introgressed inbreds and reconstituted hybrids possessed similar grain yield potential at par with the original versions. This was attributed to the selection recurrent parent alleles of SSRs linked to various loci relevant to yield attributing-and agronomic-characteristics 29 . The high recovery of RPG was further validated by great degree of similarity for the large number of DUS characters 38 . The difference for few traits between improved and original genotypes could be due to the fixation of donor allele or combination of genes from donor and recurrent parents 22 . However, few exceptions observed for the easily distinguishable morphological characteristics could be useful in registration and seed certification to differentiate newly derived genotypes from the original versions 39 .
Amylose is a linear homopolymer of glucopyranose units linked by α-(1,4) linkage, whereas amylopectin is a branched homopolymer of glucopyranose with both α-(1,4) and α-(1,6) linkages 9 . Introgressed inbreds and reconstituted hybrids recorded ~ 40% increase in amylopectin over original genotypes. Qi et al. 40 also reported ~ 23% increase in amylopectin among waxy lines and hybrids (94.9%) compared to wild type genotypes (76.9%). Accumulation of higher amylopectin in waxy landraces and hybrids have also been reported by Stamp et al. 6 Maize starch is composed of amylose and amylopectin fractions 3 . In maize, wild type Wx1 codes functionally active GBSS-I that catalyzes the formation of amylose from ADP-glucose 41 . However, recessive wx1 leads to impaired activity of GBSS-I which shifts the flux towards synthesis of amylopectin 32 . Mutant wx1 results from various types of mutations including transposon/retroposon insertion and nucleotide deletion 42 . These mutations cause formation of premature stop codon or a change in amino acids in a critical region of the transcript, as well as splicing and translational mistakes 11 . Though MABB-derived wx1-based inbreds and reconstituted hybrids recorded enhanced amylopectin, they also exhibited moderate variation in amylopectin (95-99%) despite the presence of the identical wx1 gene. This difference could be attributed to modifier loci or QTL that influence the accumulation of amylopectin in maize 9 . However, total starch content remained nearly same among the MABB-derived genotypes over their original versions. This suggested that increase in amylopectin among the www.nature.com/scientificreports/ wx1-based genotypes did not pose any negative effect on total starch content, which further justified the similar grain potential among the MAS-derived and their respective original versions. MABB-derived lines and reconstituted hybrids having o2 gene possessed higher lysine and tryptophan than the traditional maize 19 . Recessive o2 leads to reduction of zein proteins (deficient in lysine and tryptophan), with a concurrent increase in non-zein proteins rich in lysine and tryptophan 43 . o2 also down regulates the synthesis of lysine ketoglutarate reductase (LKR) resulting in increased levels of free lysine 44 . Besides, it is also involved in regulation of various lysine-rich proteins and enzymes 45 . However, wx1wx1/o2o2-based MABB derived inbreds and reconstituted hybrids possessed ~ 11-17% more lysine and tryptophan over the o2o2-based original genotypes. Zhou et al. 7 introgressed o2 gene into a waxy inbred (Zhao-OP-6/O2O2), and discovered that introgressed lines had 51.6% higher lysine than the original waxy line. Yang et al. 13 also introgressed recessive opaque16 (o16) gene from QCL3024 into two Chinese waxy lines, QCL5019 and QCL5008, and found that lysine content of the pyramid lines was 20% higher than the waxy parent. Zhang et al. 11 further pyramided o2 and o16 in a waxy genetic background and found that pyramided lines (wx1wx1/o2o2/o16o16) accumulated 11% more lysine than o2o2 genotypes. Thus, stacking of wx1 and o2 provided synergistic effects on accumulation lysine and tryptophan, which would provide better nutritional quality to alleviate malnutrition. Wang et al. 19 analyzed RNA-sequencing of kernels (18th day after pollination) of wx1wx1 and o2o2/wx1wx1 inbreds, and revealed 49 differentially expressed genes (DEGs) related to mainly catalytic activity and metabolic processes. The o2 gene regulated multiple metabolic pathways related to biological processes and molecular function during waxy maize endosperm development. In o2o2/wx1wx1 line, the two genes that encode the EF-1α and LHT1 were upregulated, and the gene that encodes sulfur-rich proteins was down-regulated, leading to the elevated levels of grain lysine 19 . Zhou et al. 7 further compared wx1wx1 inbred with o2o2/wx1wx1 inbreds and concluded that o2 introgression decreased the accumulation of various zein proteins and affected other endosperm proteins related to amino acid biosynthesis, starch-protein balance, stress response and signal transduction. Further, wx1wx1/ o2o2-based inbreds and hybrids revealed moderate variation in lysine and tryptophan despite the presence of same o2 allele. This variation is due to various modifier loci including o16 that affect regulation of amino acid biosynthesis 29,46 .
Worldwide, white maize grains are preferred as human food over yellow maize 47 . White maize is also desirable in food-processing and corn-meal industries 48 . The predominance of white maize as food is due to various reasons that include cultural preference, organoleptic property and desire for the brightly coloured finished products [49][50][51] . People in East and South-East Asia also prefer white grained waxy maize 21,52 . Keeping this in view, it was important to develop white grained waxy maize hybrids, as yellow grained waxy maize is not preferred. Since, the recurrent parents were yellow in colour and donor line had white endosperm, it was possible to develop waxy inbreds and hybrids with white colour grains. The Yellow1 (Y1) gene on chromosome-6 codes for phytoene synthase (psy1), which condenses two geranyl-geranyl pyrophosphate molecules into one molecule of phytoene in the carotenoid biosynthesis pathway 53 . The dominant Y1 allele converts the step thereby leading to the synthesis of carotenoids and eventually yellow colour in the endosperm. However, the recessive y1 allele is unable to catalyse the reaction and makes the kernel devoid of any carotenoids and eventually kernels look white 34 . In BC 2 F 3 seeds borne on BC 2 F 2 ears, Y1 gene segregated in four forms viz., (i) dark yellow (Y1Y1Y1), medium yellow (Y1Y1y1), light yellow (Y1y1y1) and white (y1y1y1) in the endosperm 54 . We selected only the white kernels to raise the BC 2 F 3 progenies, and eventually develop white grained waxy hybrids.
These newly derived white waxy hybrids possess diverse usage as food and various industrial products 3 . Globally immature waxy maize ears are gaining popularity as a breakfast item. It is also widely used to improve the viscosity, freeze-thaw stability, uniformity, and appearance of the food products 55 . Due to high amylopectin content, food made from waxy maize is easily digested in the human gut 10,56 . Amylopectin powder is a preferred food after workout in gym and body building industry 8 . Further, pure amylopectin powder possesses special pasting properties, thus used as a popular ingredient in textile, adhesive and paper industries 32 . Since waxy maize starch has a higher hydrolysis rate, it has higher starch-to-ethanol conversion efficiency when used to make ethanol 57 .
Further, these white waxy hybrids are also rich in lysine and tryptophan, thus possess superior protein quality. So far, large number of QPM hybrids rich in lysine and tryptophan have been developed and commercialized worldwide 58 . But these QPM hybrids do not possess high amount of amylopectin 11 . On the other hand, several waxy landrace and hybrids have been in cultivation especially in East-and Sout-East Asian countries 4 . These waxy cultivars are poor in nutritional quality as they lack required amount of lysine and tryptophan 13 . Though few studies have improved wx1 inbreds for nutritional qualities, the present study possesses novelty on three aspects, viz.  Zhang et al. (2013) have mentioned the enhancement of only lysine, but we analyzed the effects on both lysine and tryptophan among the waxy genotypes. These two are the essential amino acids not synthesized in our body, thus possess paramount importance for growth and development in humans, (2) earlier studies have analyzed the levels of amylopectin and lysine only in inbreds, but here we combined wx1 and o2 genes in elite inbreds, and further developed and evaluated the performance of hybrids for amylopectin, lysine, tryptophan, grain yield, and agronomic performance, and (3) previous studies have combined wx1 and o2 genes in temperate background, while lines in the present study are sub-tropically adapted. These newly derived waxy hybrids with superior protein quality would help in providing the balanced diet and alleviate the malnutrition in a sustainable and cost-efficient manner 59 . These nutritious waxy hybrids are also high yielding and would help the farmers to earn livelihood. The present investigation is the first report development of waxy hybrids rich in lysine and tryptophan using accelerated-breeding strategy.

Conclusions
Waxy maize rich in amylopectin is becoming increasingly important as a source of human nutrition, livelihood, and income generation. However, their usage as a preferred food and industrial product is limited due to lack of suitable waxy hybrids. Here, we have developed four high yielding waxy hybrids rich in amylopectin. These www.nature.com/scientificreports/ waxy hybrids also possess quality protein, besides high grain yield. The improved waxy QPM hybrids developed in this study can be directly commercialized and used for human consumption. Further, the improved waxy QPM maize inbreds will serve as potential donors for the development of the lysine and tryptophan rich waxy hybrids in the breeding programmes. This is the first report of development of maize hybrids rich in amylopectin, lysine and tryptophan. www.nature.com/scientificreports/